Project Summary Non-small cell lung cancer (NSCLC) is the most common lung cancer. Current treatments for this disease remain inadequate, and novel treatment strategies are urgently needed. Squalene epoxidase (SQLE), an enzyme controlling cholesterol biosynthesis by converting squalene to oxidosqualene, is frequently overexpressed in NSCLC. High expression of this protein is associated with poor prognosis. Thus, the goal of this application is to identify new approaches to treat high SQLE-expressing NSCLC. SQLE inhibitors are currently used in clinic for treating fungal infection partially by accumulation of squalene. Strikingly, our recent genome-wide loss-of-function screen and preliminary data suggest that SQLE inhibition by knockdown enhanced the sensitivity to inhibitors targeting the DNA damage response (DDR) kinase CHK1 and its upstream factor ATR. ATR-CHK1 axis are the key component of replication stress response. Inhibition of ATR and CHK1 leads to replication fork collapse and generation of DNA double strand breaks (DSBs), a major DNA structure that can activate ATM kinase. Given the critical role of ATM in DSB repair and cell cycle checkpoints, the cells with inhibited ATR/CHK1 activity rely heavily on ATM for survival. Our preliminary data suggest that SQLE knockdown leads to an increase in WIP1, which is a phosphatase that suppresses ATM activity. Since it has been reported previously that squalene accumulation lead to increase in WIP1 protein expression, we hypothesize that SQLE inhibition suppresses ATM activity, thereby rendering the cells sensitive to ATR and CHK1 inhibitors. Thus, a subset of NSCLC cells expressing high SQLE can be specifically targeted by the combined inhibition of SQLE and ATR or CHK1. Two Specific Aims are proposed, which are to determine (1) the mechanisms by which SQLE inhibition potentiates NSCLC cell sensitivity to ATR and CHK1 inhibitors and (2) the synergistic antitumor efficacy of combined inhibition of SQLE and ATR or CHK1. In Aim 1, we will determine whether SQLE inhibition suppresses ATM activity, thereby leading to impaired DDR, including DSB repair and cell cycle checkpoint, in a manner dependent on WIP1 and squalene. An in vitro kinase assay, DSB repair reporters, a cytogenetic assay and cell biological techniques will be used. To determine the involvement of WIP1 and squalene, SQLE inhibition-induced defects in ATM activity and subsequent DDR will be evaluated in cells expressing wild type and inactivated WIP1, and in cells with or without with squalene syntheses inhibition. In Aim 2, we will assess the antitumor efficacy of the combined inhibition of SQLE and ATR or CHK1 using in vitro assays and cell line-based and patient-derived xenograft (PDX) models. If successful, our studies will have a significant impact on improving the survival of lung cancer patients by identifying novel therapeutic approaches from the perspective of simultaneously inhibiting the proteins required for cholesterol biosynthesis and DDR.